Iron is an essential element required for growth and survival of almost every organism. Red blood cells (RBC) contain hemoglobin (Hb), a red, iron-rich protein that carries oxygen from the lungs to all of the body's muscles and organs where it reacts to provide the energy the body needs for its normal activities. When the number of red blood cells or the amount of hemoglobin they contain fall below normal, the body receives less oxygen and generates less energy than it needs to function properly. This condition in general is referred to as anemia. A common cause for anemia among infants and children is an iron deficiency. As many as 20% of children in the United States and 80% of children in developing countries will become anemic at some point by the age of 18 years. Martin, P. L., et al. The Anemias, Principles and Practices of Pediatrics, 1657 (2d ed., Lippincott 1994).
In mammals, the iron balance is primarily regulated at the level of duodenal absorption of dietary iron. In humans, hereditary hemochromatosis (HH) is a common autosomal recessive genetic disease caused by hyperabsorption of dietary iron leading to an iron overload in plasma and multiple organs, including in particular the pancreas, liver, and skin, and resulting in damages in these organs and tissues due to the iron deposits.
Juvenile hemochromatosis is an iron overload disorder caused by mutations in the gene encoding the major iron regulatory hormone hepcidin (HAMP) and hemojuvelin (HFE2). (Roetto, A., et al. 2003. Nut. Genet. 33:21-22; Papanikolaou, G., et al. 2004. Nut. Genet. 36:77-82.) It has been shown that hemojuvelin is a bone morphogenetic protein (BMP) co-receptor and that hemojuvelin-mediated BMP signals regulate hepcidin expression and iron metabolism. (Babitt, J. L., et al. 2006. Nat. Genet. 38:531-539; Babitt, J. L., et al. 2007. J Clin Invest. 117:1933-1939.) However, the endogenous BMP regulator(s) of hepcidin in vivo is unknown.
BMPs are members of the TGF-β superfamily, which is comprised of over 40 ligands. (Shi, Y., and Massague, J. 2003. Cell. 113:685-700.) These growth factors mediate diverse biological processes including cell proliferation, differentiation, apoptosis, and patterning. BMP/TGF-β superfamily ligands initiate an intracellular signaling cascade by binding to a complex of type I and type II serine threonine kinase receptors. The activated receptor complex phosphorylates intracellular Smad proteins, which then translocate to the nucleus to modulate gene expression.
Recently, a role for the BMP signaling pathway in regulating the major iron regulatory hormone hepcidin has been discovered. (Babitt, J. L., et al. 2006. Nat. Genet. 38:531-539; Babitt, J. L., H et al. J Clin Invest. 117:1933-1939; Wang, R. H., et al. 2005. Cell Metab. 2:399-409.) Secreted by the liver, hepcidin inhibits intestinal iron absorption and macrophage iron release by decreasing cell surface expression of the iron exporter ferroportin. (Nemeth, E., et al. 2004. Science. 306:2090-2093). Hepcidin is upregulated by iron administration (Pigeon, C., et al. 2001. J. Biol. Chem. 276:7811-7819, Nicolas, G., et al. 2002. J. Clin. Invest. 110:1037-1044; Nemeth, E., et al. 2004. J. Clin. Invest. 113: 1271-1276.) and inhibited by anemia. (Nicolas, G., et al. 2002. J. Clin. Invest. 110:1037-1044) Hepcidin deficiency and unchecked ferroportin activity are the common pathogenic mechanisms underlying the genetic iron overload disorder hereditary hemochromatosis due to mutations in HAMP itself, HFE2, HFE, TFR2 (encoding transferrin receptor type 2), and rare mutations of SCLAOA1 (encoding ferroportin). (Pietrangelo, A. 2006. Biochim Biophys Acta 1763:700-710) Hepcidin is also upregulated by inflammatory cytokines, such as IL-6, and hepcidin excess is implicated in the pathogenesis of anemia of inflammation (Pigeon, C., et al. 2001. J. Biol. Chem. 276:7811-7819, Nicolas, G., et al. 2002. J. Clin. Invest. 110:1037-1044; Nemeth, E., et al. 2004. J. Clin. Invest. 113: 1271-1276; Nemeth, E., et al. 2003. Blood. 101:246 1-2463; Weiss, G. and Goodnough, L. T. 2005. N. Engl. J. Med. 352:1011-1023; Andrews N C. 2008. Blood. 1122 19-30.)
Reduction of hepatic BMP signaling by a liver-specific conditional knockout of the common BMP/TGF-β intracellular mediator Smad4 (Wang, R. H., et al. 2005. Cell Metab. 2:399-409.), or by mutations in HFE2 (Papanikolaou, G., et al. 2004. Nut. Genet. 36:77-82; Babitt, J. L., et al. 2006. Nat. Genet. 38:531-539; Huang, F. W., et al. J. Clin. Invest. 115:2187-2191; Niederkofler, V., Salie, R., Arber, S. 2005. J Clin Invest. 115:2180-6), which encodes the BMP co-receptor hemojuvelin, are associated with inappropriately low hepcidin expression and iron overload. BMP signals positively increase hepcidin expression at the transcriptional level in vitro. (Babitt, J. L., et al. 2006. Nat. Genet. 38:531-539; Babitt, J. L., H et al. J Clin Invest. 117:1933-1939; Wang, R. H., et al. 2005. Cell Metab. 2:399-409; Truksa, J., et al. 2006. Proc. Natl. Acad. Sci. USA. 103:10289-10293; Verga Falzacappa, M. V., et al. 2008. J Mol. Med. 86:531-40.). Iron administration in vivo increases hepatic BMP signaling (Yu, P. B., et al. 2008. Nut Chem. Biol. 4:33-41). BMP administration in vivo increases hepcidin expression and reduces serum iron. (Babitt, J. L., H et al. J Clin Invest. 117:1933-1939). Conversely, administration of soluble hemojuvelin fused to the Fc portion of human immunoglobulin Fc (HJV.Fc) in vivo, which selectively inhibits BMP-2, BMP-4, BMP-5, and BMP-6, but not BMP-7 or BMP-9, inhibits hepcidin expression, increases ferroportin expression, mobilizes reticuloendothelial cell iron stores, and increases serum iron in vivo. (Babitt, J. L., et al. 2007. J Clin Invest. 117: 1933-1939.) Administration of the non-selective small molecule BMP inhibitor Dorsomorphin also inhibits hepcidin expression and increases serum iron in vivo. (Yu, P. B., et al. 2008. Nut Chem. Biol. 4:33-41).
Hemojuvelin (also known as RGMc) is a member of the Repulsive Guidance Molecules family of proteins, including RGMa and DRAGON(RGMb), which share 50-60% amino acid identity. (Samad, T. A., et al. 2004. J. Neurosci. 24:2027-2036) Like Hemojuvelin, RGMa (Babitt, J. L., et al. 2005. J. Biol. Chem. 280:29820-29827) and DRAGON(Samad, T. A., et al. 2005. J. Biol. Chem. 280:14122-14129) also function as co-receptors for the BMP signaling pathway.
There is a need for a cost-effective and efficient method for regulating hepcidin expression and iron metabolism.